1. BMS 602/631 - LECTURE 7 Flow Cytometry: Theory
J. Paul Robinson SVM Professor of Cytomics Professor of Biomedical Engineering Purdue University
Detectors
Notes:
Material is taken from the course text: Howard M. Shapiro, Practical Flow Cytometry, 3nd edition (1994), Wiley-Liss, New York.
RFM =Slides taken from Dr. Robert Murphy
MLM – Material taken from Melamed, et al, Flow Cytometry & Sorting, Wiley-Liss, 2nd Ed.
Purdue University
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3rd Ed. Shapiro
4th Ed. Shapiro

2. Detectors
Light must be converted from photons into volts to be measured
We must select the correct detector system according to how many photons we have available
In general, we use photodiodes for forward scatter and absorption and PMTs for fluorescence and side scatter
There are now some instruments using APDs instead of PMTs

3. Silicon photodiodes
A silicon photodiode produces current when photons impinge upon it (example are solar cells)
Does not require an external power source to operate
Peak sensitivity is about 900 nm
At 900 nm the responsivity is about 0.5 amperes/watt, at 500 nm it is 0.28 A/W
Are usually operated in the photovoltaic mode (no external voltage) (alternative is photoconductive mode with a bias voltage)
Have no gain so must have external amps
quantum efficiency ()% = 100 x (electrons out/(photons in)

4. PMT
Produce current at their anodes when photons impinge upon their light-sensitive cathodes
Require external power source
Their gain is as high as 107 electrons out per photon in
Noise can be generated from thermionic emission of electrons - this is called “dark current”
If very low levels of signal are available, PMTs are often cooled to reduce heat effects
Spectral response of PMTs is determined by the composition of the photocathode
Bi-alkali PMTs have peak sensitivity at 400 nm
Multialkali PMTs extend to 750 nm
Gallium Arsenide (GaAs) cathodes operate from nm (very costly and have lower gain)

5. Signal Detection - PMTs
Secondary emission
Cathode
Anode
Photons in
Amplified
Signal
Out
End
Window
Dynodes
Requires Current on dynodes
Is light sensitive
Sensitive to specific wavelengths
Can be end`(shown) or side window PMTs

6. Types of PMTs Side Window Signal out High voltage in
Photos: J. Paul Robinson

7. Photomultiplier tubes (PMT’s)
The PMTs in an Elite. 3 PMTs are shown, the other 2 have been removed to show their positions. A diode detector is used for forward scatter and a PMT for side scatter.
The Bio-Rad Bryte cytometer uses PMTs for forward and wide angle light scatter as well as fluorescence
Photos: J. Paul Robinson

8. PMTs
High voltage regulation is critical because the relationship between the high voltage and the PMT gain is non-linear (almost logarithmic)
PMTs must be shielded from stray light and magnetic fields
Room light will destroy a PMT if connected to a power supply
There are side-window and end-window PMTs
While photodiodes are efficient, they produce too small a signal to be useful for fluorescence

9. High Voltage on PMTs The voltage on the PMT is applied to the dynodes
This increases the “sensitivity” of the PMT
A low signal will require higher voltages on the PMT to measure the signal
When the voltage is applied, the PMT is very sensitive and if exposed to light will be destroyed
Background noise on PMTs is termed “dark noise”
PMTs generally have a voltage range from volts
Changing the gain on a PMT should be linear over the gain range
Changing the voltage on the PMT is NOT a linear function of response
Older style HH – newer PMTS have HV integrated
Photos: J. Paul Robinson

10. PMT in the optical path of an Elite cytometer
Photos: J. Paul Robinson

11. Multianode PMTs
Source:

12. Multianode PMTs
Source:

13. Multianode PMT – sensitivity and uniformity
Latest
PMT
Hamamatsu 32 Ch PMT

14. Multianode PMT – gain and spectral filtering
Now a
simple
4 color
cytometer

15. Principle of Operation
US & foreign patents pending

16. Diode Vs PMT Scatter detectors are frequently diode detectors
Sample stream
Back of Elite forward scatter detector showing the preamp
Front view of Elite forward scatter detector showing the beam-dump and video camera signal collector (laser beam and sample sheath are superimposed)
Photos: J. Paul Robinson

17. Smaller, Cheaper….but noisier…
Image Source:
Image Source:

18. APD vs PMT
Source:

19. Avalanche Photodiodes (APD’s)
Combines the best features of PMTs and photodiodes
High quantum efficiency, good gain
Gain is (much less than PMTs)
Problem with high dark current
Image From:

20. APDs
Avalanche photodiodes (APDs) are silicon photodiodes with an internal gain mechanism.
As with a conventional photodiode, absorption of incident photons creates electron-hole pairs.
However, by placing a high reverse bias voltage a strong internal electric field is created, and this accelerates the electrons through the silicon crystal lattice to produce secondary electrons by impact ionization.
The resulting electron avalanche can produce gain factors up to several hundred.

21. High through-put flow cytometry
Image Source: Howard Shapiro talk

22. Animation about detectors
APD detectors mp4

23. Single Photon Avalanche Diode (SPAD_
A single-photon avalanche diode (SPAD) is a solid-state photodetector in which a photon-generated carrier (via the internal photoelectric effect) can trigger a short-duration but relatively large avalanche current. This avalanche is created through a mechanism called impact ionization, whereby carriers (electrons and/or holes) are accelerated to high kinetic energies through a large potential gradient (voltage).
SPADs, like avalanche photodiodes (APDs), exploit the incident radiation triggered avalanche current of a p–n junction when reverse biased. The fundamental difference between SPADs and APDs is that SPADs are specifically designed to operate with a reverse-bias voltage well above the breakdown voltage. This kind of operation is also called Geiger-mode in the literature (as opposed to the linear-mode for the case of an APD). This is in analogy with the Geiger counter.
Excelitas
Miftek

26. APD vs. SiPM : -high QE and dynamic range is APD advantage
-typical gain is x100
Hamamatsu APD:
20uA/uW at 600nm PDE= 15.0 %
10uA/uW at 800nm PDE= 31.0%
Photo responsibility includes internal gain.
SiPM gain is 3E+6
APD is x10 PDE at 800nm than PMT, NUV SiPM
APD and SiPM has temperature dependence

27. Photon detection requires Electron Avalanche Effect
PMT
μPMT
Silicon Photomultilier (SiPM)
Photocurrent
Photon p n - + PD
Gain=1 -
APD Linear
Gain=100
SiPM
Geiger mode - + - + - + - + + R
Reverse Voltage
Reverse Bias Voltage

28. CCDs
Charge Coupled devices (CCD) usually in our video cameras (also called charged transfer devices)
light causes accumulation of electric charge in individual elements which release the charge at regular intervals
Useful in imaging because they can integrate over time
Not fast enough for flow cytometry application in general

29. Summary….
Photodiodes can operate in two modes - photovoltaic and photoconductive
Photodiodes are usually used for scatter
Photodiodes are more sensitive than PMTs but because of their low gain, they are not as useful for low level signals (too much noise)
PMTs are usually used for fluorescence measurements
PMTS are sensitive to different wavelengths according to the construction of the photocathode
PMTs are subject to dark current
High Voltages are not linear across the entire range

30. Lecture Summary (cont)
There is a very small time scale for measurements
Most fluorescence detectors are PMTs
PMTs can be destroyed if they receive a lot of light when powered
Standard PMTs do not have good sensitivity over 650 nm – you must use a multi-alkali PMT
New versions of Multanode PMTs are now available up to 880nm
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